Marta Lomnytska

Increased expression of cSHMT, Tbx3 and utrophin in plasma of ovarian and breast cancer patients

Plasma samples of ovarian and breast cancer patients were used to search for markers of cancer using 2‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionization time of flight mass spectrometry. Truncated forms of cytosolic serine hydroxymethyl transferase (cSHMT), T‐box transcription factor 3 (Tbx3) and utrophin were aberrantly expressed in samples from cancer patients as compared to samples from noncancerous cases. Aberrant expression of proteins was validated by immunoblotting of plasma samples with specific antibodies to cSHMT, Tbx3 and utrophin. A cohort of 79 breast and 39 ovarian cancer patients and 31 individuals with noncancerous conditions was studied. We observed increased expression of truncated cSHMT, Tbx3 and utrophin in plasma samples obtained from patients at early stages of disease. Our data suggest that cSHMT, Tbx3 and utrophin can be used as components of multiparameter monitoring of ovarian and breast cancer (supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020‐7136/suppmat/index.html).

TGFbeta1/Smad3 counteracts BRCA1-dependent repair of DNA damage.

Inactivation of the BRCA1 gene has been found to confer susceptibility to early-onset familial breast and ovarian cancers. BRCA1 regulates DNA repair, chromatin remodeling and affects gene transcription. Transforming growth factor-β (TGFβ) is a potent regulator of growth, apoptosis and invasiveness of tumor cells, including breast cancer cells. Here we show that Smad3 which is a component of the TGFβ signaling pathway, forms a complex with BRCA1 in vitro and in vivo. The interaction is mediated by the MH1 domain of Smad3 and the C-terminal part of BRCA1. We observed a co-localization of Smad3 and BRCA1 in nuclear complexes. We also found that TGFβ1/Smad3 counteracted BRCA1-dependent repair of DNA double-strand breaks in human breast epithelial cells, as evaluated by BRCA1 nuclear foci formation, single-cell gel electrophoresis and cell survival assays. Thus, TGFβ1/Smad3 suppresses BRCA1-dependent DNA repair in response to a DNA damaging agent.

Proteomics‐based identification of proteins interacting with Smad3: SREBP‐2 forms a complex with Smad3 and inhibits its transcriptional activity

Smad3 is an important component of transforming growth factor‐β (TGFβ) intracellular signalling. To identify novel interacting proteins of Smad3, we performed pull‐down assays with Smad3 constructs fused to glutathione‐S‐transferase. Proteins which formed complexes with these constructs were analyzed by two‐dimensional gel electrophoresis, and were identified by matrix‐assisted laser desorption‐ionization time‐of‐flight mass spectrometry. We identified 14 proteins interacting with the Smad3 construct lacking the N‐terminal Mad homology domain 1 (MH1), and 12 proteins interacting with the construct lacking the C‐terminal MH2 domain. Proteins involved in signalling processes, in metabolism regulation, novel proteins, and components of cytoskeleton form four groups of interacting proteins. Interactions of AGP7, sex‐determining region Y protein, actin β and sterol regulatory element binding protein‐2 (SREBP‐2) proteins with Smad3 constructs were confirmed by immunoblotting with specific antibodies. Interaction of Smad3 with SREBP‐2 was also confirmed by co‐immunoprecipitation of myc‐Smad3 and Flag‐SREBP‐2 upon expression in mammalian cells. We found that SREBP‐2 inhibited the transcriptional activity of Smad3 in luciferase reporter assays.

Differential tissue-specific protein markers of vaginal carcinoma

The objective was to identify proteins differentially expressed in vaginal cancer to elucidate relevant cancer-related proteins. A total of 16 fresh-frozen tissue biopsies, consisting of 5 biopsies from normal vaginal epithelium, 6 from primary vaginal carcinomas and 5 from primary cervical carcinomas, were analysed using two-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry. Of the 43 proteins identified with significant alterations in protein expression between non-tumourous and tumourous tissue, 26 were upregulated and 17 were downregulated. Some were similarly altered in vaginal and cervical carcinoma, including cytoskeletal proteins, tumour suppressor proteins, oncoproteins implicated in apoptosis and proteins in the ubiquitin–proteasome pathway. Three proteins were uniquely altered in vaginal carcinoma (DDX48, erbB3-binding protein and biliverdin reductase) and five in cervical carcinoma (peroxiredoxin 2, annexin A2, sarcomeric tropomyosin kappa, human ribonuclease inhibitor and prolyl-4-hydrolase beta). The identified proteins imply involvement of multiple different cellular pathways in the carcinogenesis of vaginal carcinoma. Similar protein alterations were found between vaginal and cervical carcinoma suggesting common tumourigenesis. However, the expression level of some of these proteins markedly differs among the three tissue specimens indicating that they might be useful molecular markers.

The protein kinase SIK downregulates the polarity protein Par3

The multifunctional cytokine transforming growth factor β (TGFβ) controls homeostasis and disease during embryonic and adult life. TGFβ alters epithelial cell differentiation by inducing epithelial-mesenchymal transition (EMT), which involves downregulation of several cell-cell junctional constituents. Little is understood about the mechanism of tight junction disassembly by TGFβ. We found that one of the newly identified gene targets of TGFβ, encoding the serine/threonine kinase salt-inducible kinase 1 (SIK), controls tight junction dynamics. We provide bioinformatic and biochemical evidence that SIK can potentially phosphorylate the polarity complex protein Par3, an established regulator of tight junction assembly. SIK associates with Par3, and induces degradation of Par3 that can be prevented by proteasomal and lysosomal inhibition or by mutation of Ser885, a putative phosphorylation site on Par3. Functionally, this mechanism impacts on tight junction downregulation. Furthermore, SIK contributes to the loss of epithelial polarity and examination of advanced and invasive human cancers of diverse origin displayed high levels of SIK expression and a corresponding low expression of Par3 protein. High SIK mRNA expression also correlates with lower chance for survival in various carcinomas. In specific human breast cancer samples, aneuploidy of tumor cells best correlated with cytoplasmic SIK distribution, and SIK expression correlated with TGFβ/Smad signaling activity and low or undetectable expression of Par3. Our model suggests that SIK can act directly on the polarity protein Par3 to regulate tight junction assembly.